1. Field of the Invention
The present invention relates to a method of spotting a probe on a solid support, a probe array and a method of manufacturing thereof, and a method of detecting a target single-stranded (ss) nucleic acid and a method of identifying a base sequence of a target ss nucleic acid using the probe array.
2. Related Background Art
As a method to determine a base sequence of a nucleic acid, detect a target nucleic acid in a sample, and identify various bacteria swiftly and accurately, proposed is the use of a probe array where one or more substances which can bind specifically to a target nucleic acid, so-called probes, are arranged on a solid support at a large number of sites. As a general method of manufacturing such probe arrays as described in EP No. 0373203B1, (1) the nucleic acid probe is synthesized on a solid support or (2) a previously synthesized probe is supplied onto a solid support. U.S. Pat. No. 5,405,783 discloses the method (1) in detail. Concerning the method (2), U.S. Pat. No. 5,601,980 and Science Vol. 270, p. 467 (1995) teach a method of arranging cDNA in an array by using a micropipet.
In the above method (1), it is not necessary to synthesize a nucleic acid probe in advance, since the nucleic acid probe is synthesized directly on a solid support. However, it is difficult to purify a probe nucleic acid synthesized on a solid support. The accuracy in determining the base sequence of a nucleic acid and in the detection of a target nucleic acid in a sample using a probe array largely depends on the correctness of the base sequence of the nucleic acid probe. For the method (1), therefore, further improvement in accuracy of a nucleic acid probe is required in order to manufacture a probe array of higher quality. In the method (2), a step of synthesizing a nucleic acid probe is required prior to the fixation of the nucleic acid probe on a solid support, but the nucleic acid probe can be purified before binding the probe to a solid support. For this reason, presently, the method (2) is considered to be more preferable than the method (1) as a method of manufacturing a probe array of high quality. However, the method (2) has a problem in the method of spotting a nucleic acid probe densely on a solid support. For example, when a probe array is used to determine the base sequence of a nucleic acid, it is preferable to arrange as many kinds of nucleic acid probes as possible on a solid support. When mutations in a gene are to be detected efficiently, it is preferable to arrange nucleic acid probes of sequences corresponding to the respective mutations on a solid support. In addition, when a target nucleic acid in a sample or to gene mutations and deletions are detected, it is desirable that the amount of the sample taken from a subject, specifically a blood sample, is a as small as possible. Thus, it is preferable that as much information as possible on the base sequence is obtained using a small sample amount. Considering these points, it is preferable that, for example, 10,000 or more nucleic acid probe spots per square inch are arranged in a probe array.
As the result of the research carried out by the inventors to solve above-discussed problems, they have found that an ink jet ejection method enables spotting of a probe in a markedly high density and achieved the present invention.
It is an object of the present invention to provide a method of spotting an extremely small amount of probe efficiently and accurately on a solid support without damaging the probe.
It is another object of the present invention to provide a probe array that can provide more information on nucleic acid more accurately even using a small amount of sample.
It is still another object of the present invention to provide a method of efficiently manufacturing a probe array, in which a large number of probes are bound to a solid support, with out damaging the probes.
It is further another object of the present invention to provide a method of efficiently detecting a target substance that may be contained in a sample.
It is still other object of the present invention to provide a method of identifying the structure of a target substance to obtain information on the structure of the target substance even from a small amount of sample.
According to one aspect of the present invention, there provided is a method of spotting a probe which can bind specifically to a target to a solid support. The method comprises a step of supplying a liquid containing a probe on a surface of a solid support by an ink jet method and adhering the liquid on the surface of the solid support. The use of the spotting method according to the above embodiment allows accurate and efficient provision of a probe on a solid support and efficient manufacturing of a probe array.
According to another aspect of the present invention, provided is a probe array comprised of a plurality of spots of a probe, where the spots are provided independently at a plurality of sites of the surface of a solid support in a density of 10,000 spots per square inch or higher. This probe array has spots in a remarkably high density so that much information can be obtained even from a small amount of sample.
According to further aspect of the present invention, provided is a method of manufacturing a probe array having a plurality of spots arranged independently in a plurality of sites on a surface of a solid support, the spots containing a probe which can bind specifically to a target substance comprising
a step of supplying a liquid containing the probe and attaching the liquid to a predetermined site on the surface of the solid support by means of an ink jet method. According to this embodiment, a probe array comprising spots arranged in a high density can be efficiently manufactured without damaging the probe.
According to further aspect of the present invention, provided is a method of detecting a target substance by contacting a sample with each spot of a probe array having a probe that can bind specifically to a target substance that may be contained in a sample as a plurality of independent spots on a solid support to detect a reaction product of the target substance and the probe on the solid support to detect the presence/absence of the target substance in the sample wherein the respective spots are formed by spotting a liquid containing the probe on the solid support by the ink jet method. According to this embodiment, a target substance can be detected efficiently.
According to further aspect of the present invention, provided is a method of identifying a structure of a target substance contained in a sample comprising:
a step of preparing a probe array provided with spots of a probe, which can bind specifically to a specific substance, on a surface of a solid support;
a step of contacting the sample to the spots; and
a step of detecting binding between the target substance and the probe.
U.S. Pat. No. 5,601,980 states that it is inappropriate to use a conventional ink jet method in spotting of a nucleic acid probe. In lines 31-52 in the second column of U.S. Pat. No. 5,601,980, it is said that the use of the ink jet printer technique in which a small amount of ink is ejected by pressure wave is inappropriate, because the pressure wave for ejecting ink may lead to a drastic rise in the ink temperature and damage the nucleic acid probe and scattering of the ink upon ejection may lead to contamination of adjacent probe spots. Considering this, U.S. Pat. No. 5,601,980 discloses a method of manufacturing a probe array in which a drop of a liquid containing a nucleic acid probe is formed on a tip of a micropipet utilizing gas pressure, while monitoring the size of the drop, application of pressure is terminated when the drop becomes the predetermined size, and the drop is applied on a solid support.
U.S. Pat. No. 5,474,796 discloses manufacturing of oligonucleotide array by forming a matrix of hydrophobic and hydrophilic parts on a solid support surface and ejecting four nucleotides sequentially to the hydrophilic part by means of a piezoelectric impulse jet pump apparatus and a method of determination of the base sequence of a target nucleic acid using the oligonucleotide array. However, these prior arts do not disclose a method in which nucleic acid probes each having a base sequence of a predetermined length is ejected in advance using an ink jet technique to arrange the nucleic acid probes accurately and densely.